Biology Reference
In-Depth Information
of medium containing 10, 100, 500, or 1,000 μg/ml of lipids. Upon 24 hr at 37°C, the
cells were washed with 0.1 M phosphate buffered saline (PBS) pH 7.2, and incubated
with 200 μg/ml of XTT for 4 hr. The XTT solution was removed and the extent of
reduction of XTT to formazan within the cells was quantifi ed by measuring the absor-
bance at 450 nm using an ELISA reader. Liposomes made of HSPC:cholesterol (1:1,
M:M) were used as control.
Cell Uptake
Cell uptake and intracellular fate of ARC loaded with the fluorophore/quencher pair
HPTS/DPX was followed upon incubation with J-774 macrophages by fluorescence
microscopy. The ARC loaded with HPTS/DPX was prepared as stated before, except
that the lipid films were hydrated with a solution of 35 mM HPTS and 50 mM DPX
in Tris buffer. The J-774 cells grown to near confluence on rounded coverslips in 24-
well plate were incubated with 10 μg/ml μg of ARC-
GC
/
BM
-HPTS/DPX at 37°C for
10, 20, 30, 45, and 60 min. After incubation, suspensions were removed, cells were
washed, and coverslips were mounted on a fluorescence microscope. Cell-associated
HPTS fluorescence was monitored using a Nikon Alphaphot 2 YS2 instrument.
Similarly, the intracellular fate of both fl uorescent ARC upon 45 min-incubation
with J-774 cells was followed by monitoring the HPTS fl uorescent signal along 1 hr.
Immunization
Female 6-8-week-old C3H/HeN mice were obtained from University of Buenos Ai-
res, Argentina, and maintained under standard conditions. The study was conducted
following the Institutional Experimental Guidelines for Animal Studies. Groups of
five animals were immunized sc on days 0 and 21 with 25 μg of BSA; 25 μg of
BSA with added 100 μg of Al
2
O
3
(BSA-Al, Alhydrogel
®
, Superfos Biosector, Ved-
baek, Denmark) or 25 μg of BSA entrapped in any type of ARC (ARC-
GC
-BSA or
ARC-
BM
-BSA; 1.3 mg of archaeal lipids each ARC). Control mice were injected with
equivalent amount of empty TEB. All groups were boosted with 25 μg of adjuvant-
free BSA on day 180.
Evaluation of Antibody Response
Blood was collected from the tail vein at various time-points after immunization, as
specified in the figure legends, and sera were analyzed by ELISA for the presence
of anti-BSA antibodies. Briefly, microtiter plates (Nunc, Roskilde, Denmark) were
coated overnight at 4°C with 45 μg/ml BSA diluted in 0.1 M carbonate-bicarbonate
buffer (pH 9.6) and then blocked for 1 hr at 37°C with PBS containing 0.2% Tween
20 (0.2% PBST) after washing with 0.05% PBST. Another wash as above described
was followed by the addition of 100 μl of 3-fold dilutions of individual sera in 0.05%
PBST. After 2 hr at 37°C and further washing, the plates were incubated for 1 hr at
37°C with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce, Rockford,
IL) diluted 1:2000 in 0.025% PBST. For antibody isotyping, horseradish peroxidase-
conjugated rat anti-mouse IgG1 or IgG2a revealing antisera (PharMingen, San Diego,
CA), diluted 1:1,000, were used. The plates were further washed and the reactions
were developed by adding the ABTS substrate (2, 2'-azino-bis (3-ethylbenzthiazoline-
